1. Field of the Invention
The present invention is directed to a method for separating phosphopeptides from ATP which is useful for detecting protein kinase activities and specifically, to a chromatographic column method in which a stopped reaction mixture containing phosphopeptides and ATP is passed through a column containing a cation exchange resin and then through another column containing an anion exchange resin, and the eluate which contains phosphopeptides free from ATP is then recovered.
2. Description of Related Art
Protein kinases are a large class of biologically important molecules. Protein kinase activities are generally assayed by measuring the transfer of phosphate from [.gamma.-.sup.32 P]ATP to a substrate. The sensitivity of the assay relies on effective separation of the radiolabeled product from ATP. With a protein as the substrate, the phosphoprotein may be precipitated with acid, redissolved in base to remove trapped ATP, (D. A. Walsh et al, (1971) J. Biol. Chem. 246, 1977-1985), followed by reprecipitation with acid and trapping on paper filter disks, (E. M. Reimann et al, (1971) J. Biol. Chem. 246, 1986-1995), glass fiber filters, (J. Erlichman et al, (1971) Proc. Natl. Acad. Sci. USA 68, 731-735), or cellulose acetate filters (J. L. Goldstein et al (1973) J. Biol. Chem. 248, 6300-6307). Synthetic peptides have also been employed as protein kinase substrates. With the use of an anion exchange resin, one may achieve quantitative recovery of a phosphopeptide and effective separation of the phosphopeptide from the radioactive ATP (G. Tessmer et al (1973) Biochem. Biophys. Res. Commun. 50, 1-7; and B. E. Kemp et al, (1976) Proc. Natl. Acad. Sci. USA 73, 1038-1042). Another method involves trapping of phosphoproteins (J. J. Witt et al,. (1975) Anal. Biochem. 66, 253- 258) and phosphopeptides (D. B. Glass et al, (1978) Anal. Biochem. 87, 566-575) on phosphocellulose paper under acidic conditions. ATP is removed more effectively with this phosphocellulose method in the presence of phosphoric acid (R. Roskoski (1983) in Methods in Enzymology (J. D. Corbin et al, Eds.), Vol. 99, pp. 3-6, Academic Press, New York).
Although the above procedures are well established for measuring protein kinase activities, all have certain drawbacks. First, the typical background in any of the published procedures is 0.04 to 0.1% of the initial radioactivity. Thus, under typical protein kinase assay conditions, with 1,000,000 cpm of [.gamma.-.sup.32 P]ATP in the reaction mixture, the background radioactivity in assay blanks is 400 to 1000 cpm of .sup.32 P. It is this background that determines sensitivity and dictates the amount of radioactive substrate and enzyme required for the detection of protein kinase activity. Second, all of the methods are somewhat tedious. It is therefore desirable to reduce both assay background and labor involved in assaying protein kinases.